Novel assay

ABSTRACT

This invention relates to a method for identification of an agent that modulates activity of G-protein coupled receptor 41 (GPR 41), or G-protein coupled receptor 42 (GPR 42) which method comprises: (i) contacting a test agent with GPR 41, GPR42 or a variant of either thereof which is capable of coupling to a G-protein; and (ii) monitoring for GPR 41 or GPR 42 activity in the presence of a G-protein; thereby determining whether the test agent modulates GPR 41 or GPR 42 activity. An agent identifiable by this method is provided for use in the treatment of dyslipidaemia, coronary heart disease, atheroselerosis, thrombosis or obesity, angina, chronic renal failure, peripheral vascular disease, stroke, type II diabetes or metabolic syndrome (syndrome X).

FIELD OF THE INVENTION

[0001] The present invention relates to the identification of modulatorsof G-protein coupled receptors, and the use of such modulators in thetreatment of adipocyte associated conditions.

BACKGROUND OF THE INVENTION

[0002] G-protein coupled receptors (GPCRs) are a super-family ofmembrane receptors that mediate a wide variety of biological functions.Upon binding of extracellular ligands, GPCRs interact with a specificsubset of heterotrimeric G proteins that can, in their activated forms,inhibit or activate various effector enzymes and/or ion channels. AllGPCRs are predicted to share a common molecular architecture consistingof seven transmembrane helices linked by alternating intracellular andextracellular loops. The extracellular receptor surface has been shownto be involved in ligand binding whereas the intracellular portions areinvolved in G protein recognition and activation.

[0003] Activation of receptors coupled to the G_(i) family of G proteinsleads to inhibition of adenylate cyclase and lowering of intracellularcAMP levels. In adipocytes this leads to inhibition of hormone-sensitivelipase (HSL) which regulates the process of lipolysis, i.e. thehydrolysis of triglycerides (TG) to glycerol and non-esterified fattyacids (NEFA). Inhibition of lipolysis and the concomitant lowering ofNEFA levels cause a reduction of hepatic triglyceride synthesisresulting in a fall in the levels of TG-rich lipoproteins. This thenleads to an elevation in high-density lipoprotein (HDL) levels, thusgiving the desired clinical profile of high HDL and low TG for thetreatment of dyslipidemia.

[0004] Furthermore, there are many epidemiological studies illustratingan inverse correlation between plasma HDL cholesterol and coronaryartery disease. Many patients with decreased plasma HDL cholesterollevels also have elevated TG levels. Therefore an agent that inhibitsadipocyte lipolysis, thereby reducing TG availability, may also resultin an increase in plasma HDL cholesterol levels due to the equilibriumthat exists between the levels HDL, LDL and triglycerides.

[0005] Adipocytes are known to express a number of G_(i)-coupledreceptors such as the adenosine A₁, prostaglandin EP3 and nicotinic acidreceptors. Agonists at such GPCRs have been shown to be anti-lipolytic,i.e. they promote lipid lowering, and in the case of nicotinic acid havebeen used in the clinic to treat particular forms of dyslipidaemia.However, unlike the adenosine A₁ and EP3 receptors, the nicotinic acidreceptor has yet to be identified at the molecular level.

SUMMARY OF THE INVENTION

[0006] The present invention is based on the finding that expression ofthe G-protein coupled receptors GPR 41 and GPR 42 is restricted toadipose tissue. GPR 41 or GPR 42 may therefore be used as a screeningtarget for the identification and development of novel pharmaceuticalagents for use inhibiting lipolysis. Accordingly the present inventionprovides a method for identification of an agent that modulates GPR 41or GPR 42 activity, which method comprises:

[0007] (i) contacting a test agent with a cell, such as an adipocyte,which expresses GPR 41, GPR 42 or a variant of either thereof which iscapable of coupling to a G-protein; and

[0008] (ii) monitoring for GPR 41 or GPR 42 activity in the presence ofa G-protein;

[0009] thereby determining whether the test agent modulates GPR 41 orGPR 42 activity.

[0010] The test agent may be contacted in step (i) with cells thatexpress GPR 41, GPR 42 or a variant of either thereof. Alternatively,the test agent may be contacted in step (i) with membrane obtained fromsuch cells. The invention also provides:

[0011] a test kit suitable for identification of an agent that modulatesGPR 41 or GPR 42 activity, which kit comprises:

[0012] (a) GPR 41, GPR 42 or a variant of either thereof which iscapable of coupling to a G-protein; and

[0013] (b) means for monitoring GPR 41 or GPR 42 activity.

[0014] a method for identification of an agent that inhibits lipolysis,which method comprises contacting adipocytes in vitro with a test agentwhich modulates GPR 41 or GPR 42 activity and which has been identifiedby the method of the invention and monitoring lipolysis, therebydetermining whether the test substance is an inhibitor of lipolysis;

[0015] an activator of GPR 41 or GPR 42 activity or an inhibitor oflipolysis identified by a method of the invention or a polynucleotidewhich encodes GPR 41, GPR 42 or a variant polypeptide of either thereof,for use in a method of treatment of the human or animal body by therapy;and

[0016] use of such an activator, inhibitor or polynucleotide in themanufacture of a medicament for the treatment of dyslipidaemia andconditions associated with dyslipidaemia, coronary heart disease,atheroselerosis, thrombosis or obesity, angina, chronic renal failure,peripheral vascular disease, stroke, type II diabetes or metabolicsyndrome (syndrome X).

[0017] The polynucleotide may comprise:

[0018] (a) the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 3,

[0019] (b) a sequence which hybridizes under stringent conditions to thecomplement of SEQ ID NO: 1 or SEQ ID NO: 3,

[0020] (c) a sequence that is degenerate as a result of the genetic codewith respect to a sequence defined in (a) or (b), or

[0021] (d) a sequence having at least 60% identity to a sequence asdefined (a), (b) or (c).

BRIEF DESCRIPTION OF THE FIGURES

[0022]FIG. 1 illustrates the expression of GPR 41 in normal humantissues.

[0023]FIG. 2 illustrates the effect of expression of GPR 41 on theability of acetate to stimulate GTPγS binding on membranes from HEK293Tcells.

[0024]FIG. 3 illustrates the effect of transient expression of human GPR41, on carboxylic acid-mediated stimulation of GTPγS binding in HEK293Tcells.

[0025]FIG. 4 illustrates the stimulatory effect of 3-hydroxybutyrate onGTPγS binding in HEK293T cell membranes transfected to express GPR41/G_(o1α).

[0026]FIG. 5 illustrates the effect of transient expression of rat GPR41 on carboxylic acid-mediated stimulation of GTPγS binding in HEK293Tcells.

[0027]FIG. 6 illustrates the coupling of rat GPR 41 to yeast pheremoneresponse pathways via G protein chimeras.

[0028]FIG. 7 illustrates the effect of various doses of propionate onrat GPR 41 expressed in Saccharomyces cerevisiae.

[0029]FIG. 8 illustrates the effect of carboxylic acid on rat GPR 41expressed in Saccharomyces cerevisiae.

[0030]FIG. 9 illustrates the effect of 3-hydroxybutyrate on rat GPR 41expressed in Saccharomyces cerevisiae.

[0031]FIG. 10 illustrates the effect of carboxylic acid on lipolysis inrat primary adipocytes.

[0032]FIG. 11 illustrates the effect of sodium acetate onisoprenaline-stimulated adenylate cyclase activity in rat primaryadipocytes.

[0033]FIG. 12 illustrates the expression of G protein coupled receptor42 (GPR 42) in normal human tissues.

BRIEF DESCRIPTION OF THE SEQUENCES

[0034] SEQ ID NO: 1 shows the DNA and amino acid sequences of human GPR41.

[0035] SEQ ID NO: 2 is the amino acid sequence alone of GPR 41. Theseven transmembrane domains are identified.

[0036] SEQ ID NO: 3 shows the DNA and amino acid sequences of human GPR42.

[0037] SEQ ID NO: 4 shows the amino acid sequence alone of GPR 42.

[0038] SEQ ID NO: 5 shows the DNA and amino acid sequence of rat GPR 41.

[0039] SEQ ID NO: 6 shows the amino acid sequence alone of rat GPR 41.

[0040] SEQ ID NO: 7 shows the sequence of PCR primer NF415.

[0041] SEQ ID NO: 8 shows the sequence of PCR primer NF416.

[0042] SEQ ID NO: 9 shows the sequence of PCR primer NF417.

[0043] SEQ ID NO: 10 shows the sequence of PCR primer NF412.

[0044] SEQ ID NO: 11 shows the sequence of PCR primer NF419.

[0045] SEQ ID NO: 12 shows the sequence of PCR primer NF420.

DETAILED DESCRIPTION OF THE INVENTION

[0046] Throughout the present specification and the accompanying claimsthe words “comprise” and “include” and variations such as “comprises”,“comprising”, “includes” and “including” are to be interpretedinclusively. That is, these words are intended to convey the possibleinclusion of other elements or integers not specifically recited, wherethe context allows.

[0047] The present invention relates to human G-protein coupledreceptors, GPR 41, GPR 42 and variants of either thereof. G-proteincoupled receptors GPR 41 and GPR 42 are closely related. GPR 41 and GPR42 have been cloned previously (Sawzdargo et al, Biochem. Biophys. Res.Commun. 239, 543-547, 1997). Sequence information for GPR 41 is providedin SEQ ID NO: 1 (nucleotide and amino acid) and in SEQ ID NO: 2 (aminoacid). Similarly sequence information for GPR 42 is provided in SEQ IDNO: 3 (nucleotide and amino acid) and in SEQ ID NO: 4 (amino acid).Sequence information for rat GPR 41 is provided in SEQ ID NO: 5(nucleotide and amino acid) and in SEQ ID NO: 6 (amino acid). Theinvention can therefore use polypeptides consisting essentially of theamino acid sequence of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6 or afunctional variant of either sequence. A functional chimeric receptorcontaining a fragment of SEQ ID NO: 2 or SEQ ID NO: 4 may therefore beused.

[0048] The term “variant” refers to a polypeptide which has the sameessential character or basic biological functionality as GPR 41 or GPR42. The essential character of GPR 41 and GPR 42 can be defined as thatof a G-protein coupled receptor. Both GPR 41 and GPR 42 couple toG_(i)-protein. Thus, the term “variant” refers in particular to apolypeptide which activates G_(i).

[0049] To determine whether a candidate variant has the same function asGPR 41 or GPR 42, the ability of the variant to activate G_(i)-proteincan be determined. The effect of the candidate variant on G_(i)activation can be monitored. This can be carried out, for example, bycontacting cells expressing the candidate variant with a ligand whichactivates G_(i)-protein when contacted with cells that express GPR 41 orGPR 42, and measuring a G_(i)-coupled readout. A control experiment istypically also carried out in which cells of the same type as thoseexpressing the candidate variant, but expressing GPR 41 or GPR 42instead, are contacted with the ligand and a corresponding G_(i)-coupledreadout is measured. The effect attained by the candidate variant canthen be directly compared with that attained by GPR 41 or GPR 42.

[0050] An alternative way to determine whether a variant polypeptide hasthe same function as GPR 41 or GPR 42 is to determine whether thevariant polypeptide binds to a ligand which activates G_(i) when theligand is contacted with GPR 41 or GPR 42. Thus, the ligand shouldactivate G_(i) when contacted with cells that express GPR 41 or GPR 42.The ability of a candidate variant to bind such a ligand can bedetermined directly by contacting the candidate variant with aradiolabelled ligand that binds to GPR 41 or GPR 42 and monitoringbinding of the ligand to the variant. Typically, the radiolabelledligand can be incubated with cell membranes containing the candidatevariant. The membranes can then be separated from non-bound ligand anddissolved in scintillation fluid to allow the radioactivity of themembranes to be determined by scintillation counting. Non-specificbinding of the candidate variant may also be determined by repeating theexperiment in the presence of a saturating concentration ofnon-radioactive ligand. Preferably a binding curve is constructed byrepeating the experiment with various concentrations of the candidatevariant. The ability to bind a ligand of GPR 41 or GPR 42 may also bedetermined indirectly as described below.

[0051] Typically, polypeptides with more than about 65% identity,preferably at least 80% or at least 90% and particularly preferably atleast 95%, at least 97% or at least 99% identity, with the amino acidsequence of SEQ ID NO: 1, 2, 3 or 4 over a region of at least 20,preferably at least 30, at least 40, at least 60 or at least 100contiguous amino acids or over the full length of the amino acidsequence of SEQ ID NO: 1, 2, 3 or 4 are considered as GPR 41 or GPR 42variants. The UWGCG Package provides the BESTFIT program which can beused to calculate identity (for example used on its default settings)(Devereau et al (1984) Nucleic Acid Research 12, p387-395). The PILEUPand BLAST algorithms can be used to calculate identity or line upsequences (typically on their default settings), for example asdescribed in Algschul S. F. (1993) J. Mol. Evol. 36: 290-300; Altschul,S. F. et al (1990) J. Mol. Biol. 215: 403-10. Software for performingBLAST analyses is publicly available through the National Centre forBiotechnology Information (http://www.ncbi.nlm.nih.gov/).

[0052] Variant polypeptides therefore include naturally occurringallelic variants. An allelic variant will generally be of human ornon-human mammal origin, such as bovine or porcine origin.Alternatively, a variant polypeptide can be a non-naturally occurringsequence. A non-naturally occurring variant may thus be a modifiedversion of GPR 41 or GPR 42, i.e. a modified version of the polypeptidehaving the amino acid sequence of SEQ ID NO: 1,2, 3 or 4.

[0053] The amino acid sequence of GPR 41 or GPR 42 may be modified bydeletion and/or substitution and/or addition of single amino acids orgroups of amino acids as long as the modified polypeptide retains thecapability to function as a G-protein coupled receptor. Such amino acidchanges may occur in one, two or more of the intracellular domains ofGPR 41 or GPR 42 and/or one, two or more of the extracellular domains ofGPR 41 or GPR 42and/or one, two or more of the transmembrane domains ofGPR 41 or GPR 42.

[0054] Amino acid substitutions may thus be made, for example from 1, 2,3, 4 or 5 to 10, 20 or 30 substitutions. Conservative substitutions maybe made, for example according to Table 1 below. Amino acids in the sameblock in the second column and preferably in the same line in the thirdcolumn may be substituted for each other. TABLE 1 Conservative aminoacid substitutions ALIPHATIC Non-polar G A P I L V Polar-uncharged C S TM N Q Polar-charged D E K R AROMATIC H F W Y

[0055] A variant polypeptide may be a shorter polypeptide. For example,a polypeptide of at least 20 amino acids or up to 50, 60, 70, 80, 100 or150 amino acids in length may constitute a variant polypeptide as longas it demonstrates the functionality of GPR 41 or GPR 42. A variantpolypeptide may therefore lack one, two or more intracellular domainsand/or one, two or more extracellular domains and/or one, two or moretransmembrane domains. A variant polypeptide may thus be a fragment ofthe full length polypeptide. A shortened polypeptide may comprise aligand-binding region (N-terminal extracellular domain) and/or aneffector binding region (C-terminal intracellular domain). Suchfragments can be used to construct chimeric receptors preferably withanother 7-transmembrane G-coupled receptor.

[0056] Variant polypeptides include polypeptides that are chemicallymodified, e.g. post-translationally modified. For example, such variantpolypeptides may be glycosylated or comprise modified amino acidresidues. They may also be modified by the addition of histidineresidues, for example 6 or 8 His residues, or an epitope tag, forexample a T7, HA, myc or flag tag, to assist their purification ordetection. They may be modified by the addition of a signal sequence topromote insertion into the cell membrane.

[0057] The invention also utilises nucleotide sequences that encode GPR41, GPR 42 or variants of either thereof as well as nucleotide sequenceswhich are complementary thereto. The nucleotide sequence may be RNA orDNA including genomic DNA, synthetic DNA or cDNA. Preferably thenucleotide sequence is a DNA sequence and most preferably, a cDNAsequence. Nucleotide sequence information is provided in SEQ ID NO: 1and SEQ ID NO: 3. Such nucleotides can be isolated from human cells orsynthesised according to methods well known in the art, as described byway of example in Sambrook et al, Molecular Cloning: A LaboratoryManual, 2nd edition, Cold Spring Harbour Laboratory Press, 1989.Typically a useful polynucleotide comprises a contiguous sequence ofnucleotides which is capable of hybridising under selective conditionsto the coding sequence or the complement of the coding sequence of SEQID NO: 1 or SEQ ID NO: 3.

[0058] A polynucleotide can hybridize to the coding sequence or thecomplement of the coding sequence of SEQ ID NO: 1 or SEQ ID NO: 3 at alevel significantly above background. Background hybridisation mayoccur, for example, because of other cDNAs present in a cDNA library.The signal level generated by the interaction between a polynucleotideand the coding sequence or complement of the coding sequence of SEQ IDNO: 1 or SEQ ID NO: 3 is typically at least 10 fold, preferably at least100 fold, as intense as interactions between other polynucleotides andthe coding sequence of SEQ ID NO: 1 or SEQ ID NO: 3. The intensity ofinteraction may be measured, for example, by radiolabelling the probe,e.g. with ³²P. Selective hybridisation may typically be achieved usingconditions of low stringency (0.3M sodium chloride and 0.03M sodiumcitrate at about 40° C.), medium stringency (for example, 0.3M sodiumchloride and 0.03M sodium citrate at about 50° C.) or high stringency(for example, 0.03M sodium chloride and 0.003M sodium citrate at about60° C.).

[0059] The coding sequence of SEQ ID NO: 1 or SEQ ID NO: 3 may bemodified by one or more nucleotide substitutions, for example from 1, 2,3, 4 or 5 to 10, 25, 50 or 100 substitutions. The polynucleotide of SEQID NO: 1 or SEQ ID NO: 3 may alternatively or additionally be modifiedby one or more insertions and/or deletions and/or by an extension ateither or both ends. The modified polynucleotide generally encodes apolypeptide which has G-protein coupled receptor activity or inhibitsthe activity of GPR 41 or GPR 42. Degenerate substitutions may be madeand/or substitutions may be made which would result in a conservativeamino acid substitution when the modified sequence is translated, forexample as shown in the Table above.

[0060] A nucleotide sequence which is capable of selectively hybridisingto the complement of the DNA coding sequence of SEQ ID NO: 1 or SEQ IDNO: 3 will generally have at least 60%, at least 70%, at least 80%, atleast 90%, at least 95%, at least 98% or at least 99% sequence identityto the coding sequence of SEQ ID NO: 1 or SEQ ID NO: 3 respectively,over a region of at least 20, preferably at least 30, for instance atleast 40, at least 60, more preferably at least 100 contiguousnucleotides or most preferably over the full length of SEQ ID NO: 1 orSEQ ID NO: 3 respectively. Methods of measuring nucleic acid and proteinhomology are well known in the art. For example the UWGCG Packageprovides the BESTFIT program which can be used to calculate homology(Devereux et al 1984). Similarly the PILEUP and BLAST algorithms can beused to line up sequences (for example are described in Altschul 1993,and Altschul et al 1990). Many different settings are possible for suchprograms. In accordance with the invention, the default settings may beused.

[0061] Any combination of the above mentioned degrees of sequenceidentity and minimum sizes may be used to define polynucleotides of theinvention, with the more stringent combinations (i.e. higher sequenceidentity over longer lengths) being preferred. Thus, for example apolynucleotide which has at least 90% sequence identity over 25,preferably over 30 nucleotides forms one aspect of the invention, asdoes a polynucleotide which has at least 95% sequence identity over 40nucleotides.

[0062] Polynucleotides may be used as a primer, eg a PCR primer or aprimer for an alternative amplification reaction of a probe, eg labelledwith a revealing label by conventional means for identifying mutationsin GPR 41 or GPR 42 that may be implicated in diseases resulting fromabnormal lipolysis. Fragments of polynucleotides may be fused to thecoding sequence of other proteins, preferably other G-protein coupledreceptors, to form a sequence coding for a fusion protein.

[0063] Such primers, probes and other fragments will preferably be atleast 10, preferably at least 15 or at least 20, for example at least25. at least 30 or at least 40 nucleotides in length. They willtypically be up to 40, 50, 60, 70, 100 or 150 nucleotides in length.Probes and fragments can be longer than 150 nucleotides in length, forexample up to 200, 300, 400, 500 nucleotides in length, or even up to afew nucleotides, such as five or ten nucleotides, short of the codingsequence of SEQ ID NO: 1 or SEQ ID NO: 3.

[0064] The polynucleotides have utility in production of GPR 41, GPR 42or variant polypeptides, which may take place in vitro, in vivo or exvivo. The polynucleotides may be used as therapeutic agents in their ownright, in gene therapy techniques. The polynucleotides are cloned intoexpression vectors for these purposes. Such expression vectors areroutinely constructed in the art of molecular biology and may forexample involve the use of plasmid DNA and appropriate initiators,promoters, enhancers and other elements, such as for examplepolyadenylation signals which may be necessary, and which are positionedin the correct orientation, in order to allow for protein expression.Other suitable vectors would be apparent to a person skilled in the art.By way of further example in this regard we refer to Sambrook et al.

[0065] Expression vectors comprise a polynucleotide encoding the desiredpolypeptide operably linked to a control sequence which is capable ofproviding for the expression of the coding sequence by a host cell. Theterm “operably linked” refers to a juxtaposition wherein the componentsdescribed are in a relationship permitting them to function in theirintended manner. A regulatory sequence, such as a promoter, “operablylinked” to a coding sequence is positioned in such a way that expressionof the coding sequence is achieved under conditions compatible with theregulatory sequence.

[0066] The vectors may be plasmid, virus or phage vectors provided witha origin of replication, optionally a promoter for the expression of thesaid polynucleotide and optionally a regulator of the promoter. Thevectors may contain one or more selectable marker genes, for example anampicillin resistance gene in the case of a bacterial plasmid or aresistance gene for a fungal vector. Vectors may be used in vitro, forexample for the production of RNA or DNA or used to transfect ortransform a host cell, for example, a mammalian host cell. The vectorsmay also be adapted to be used in vivo, for example in a method of genetherapy.

[0067] Promoters and other expression regulation signals may be selectedto be compatible with the host cell for which expression is designed.For example, yeast promoters include S. cerevisiae GAL4 and ADHpromoters, S. pombe nmt1 and adh promoter. Mammalian promoters includethe metallothionein promoter which can be induced in response to heavymetals such as cadmium. Viral promoters such as the SV40 large T antigenpromoter or adenovirus promoters may also be used. All these promotersare readily available in the art.

[0068] Mammalian promoters, such as β-actin promoters, may be used.Tissue-specific promoters, in particular adipose cell specific promotersare especially preferred. Viral promoters may also be used, for examplethe Moloney murine leukaemia virus long terminal repeat (MMLV LTR), therous sarcoma virus (RSV) LTR promoter, the SV40 promoter, the humancytomegalovirus (CMV) IE promoter, adenovirus, HSV promoters (such asthe HSV IE promoters), or HPV promoters, particularly the HPV upstreamregulatory region (URR). Viral promoters are readily available in theart.

[0069] The vector may further include sequences flanking thepolynucleotide which comprise sequences homologous to eukaryotic genomicsequences, preferably mammalian genomic sequences, or viral genomicsequences. This will allow the introduction of the relevantpolynucleotides into the genome of eukaryotic cells or viruses byhomologous recombination. In particular, a plasmid vector comprising theexpression cassette flanked by viral sequences can be used to prepare aviral vector suitable for delivering the polynucleotides of theinvention to a mammalian cell. Retrovirus vectors for example may beused to stably integrate the polynucleotide into the host genome.Replication-defective adenovirus vectors by contrast remain episomal andtherefore allow transient expression.

[0070] Cells are transformed or transfected with the vectors to expressthe GPR 41 or GPR 42 polypeptide or a variant of either thereof. Suchcells may be eucaryotic or prokaryotic. They include transient or,preferably, stable higher eukaryotic cell lines such as mammalian cellsor insect cells, lower eukaryotic cells such as yeast, and prokaryoticcells such as bacterial cells. Particular examples of cells which may beused to express GPR 41, GPR 42 or a variant polypeptide includemammalian HEK293T, CHO, HeLa and COS7 cells. Preferably the cell lineselected will be one which is not only stable, but also allows formature glycosylation and cell surface expression of the GPR 41 or GPR 42polypeptide or a variant of either thereof. Cells such as adipocytesexpressing the GPR 41 or GPR 42 receptor or a variant polypeptide may beused in screening assays. Expression may be achieved in transformedoocytes. The GPR 41 or GPR 42 polypeptide or a variant of either thereofmay be expressed in cells such as adipose tissue of a transgenicnon-human animal, preferably a rodent such as a mouse.

[0071] The present invention is concerned in particular with the use ofGPR 41, GPR 42 or a functional variant in screening methods to identifyagents that may act as modulators of GPR 41 or GPR 42 receptor activityand, in particular, agents that may act as modulators of lipolysis. Suchmodulators are useful in the treatment of dyslipidaemia, coronary arterydisease, atherosclerosis, obesity and thrombosis, angina, chronic renalfailure, peripheral vascular disease, stroke, type II diabetes andmetabolic syndrome (syndrome X).

[0072] Any suitable form of assay may be employed to identify amodulator of GPR 41 or GPR 42 activity and/or of lipolysis. In generalterms, such screening methods involve contacting GPR 41, GPR 42 or avariant polypeptide with a test compound and then determining receptoractivity. G-protein activation, and especially G_(i)-protein activation,may be determined therefore. Where a test compound affects receptoractivity, its effect on lipolysis can be determined by contactingadipocytes in culture with the test compound and measuring lipolysis.

[0073] Modulator activity can be determined in vitro or in vivo bycontacting cells expressing GPR 41, GPR 42 or a variant polypeptide withan agent under test and by monitoring the effect mediated by the GPR 41,GPR 42 or variant polypeptide. Thus, a test agent may be contacted withisolated cells which express GPR 41, GPR 42 or a variant polypeptide.The cells may be provided in culture. Cells may be disrupted and cellmembranes isolated and used.

[0074] The GPR 41, GPR 42 or variant polypeptide may be naturally orrecombinantly expressed. Preferably, an assay is carried out in vitrousing cells expressing recombinant polypeptide or using membranes fromsuch cells. Suitable eucaryotic and procaryotic cells are discussedabove. Preferably adipocytes are used.

[0075] Typically, receptor activity is monitored by measuring aG_(i)-coupled readout. G_(i)-coupled readout can be monitored using anelectrophysiological method to determine the activity of G-proteinregulated Ca²⁺ or K⁺ channels or by using fluorescent dye to measurechanges in intracellular Ca²⁺ levels. Other methods that can typicallybe used to monitor receptor activity involved measuring levels of oractivity of GTPγS or cAMP.

[0076] A standard assay for measuring activation of the G_(i) family ofG proteins is the GTP_(γ)S binding assay. Agonist binding to Gprotein-coupled receptors promotes the exchange of GTP for GDP bound tothe α subunit of coupled heterotrimeric G proteins. Binding of thepoorly hydrolysable GTP analogue, [³⁵S]GTP_(γ)S, to membranes has beenused extensively as a functional assay to measure agonism at a widevariety of receptors. Furthermore, the assay is largely restricted tomeasuring function of receptors coupled to the G_(i) family of Gproteins due to their ability to bind and hydrolyse guanine nucleotideat significantly higher rates than members of the G_(q), G_(s) and G₁₂families. See Wieland and Jakobs, Methods Enzymol. 237, 3-13, 1994.

[0077] Yeast assays may be used to screen for agents that modulate theactivity of GPR 41, GPR 42 or variant polypeptides. A typical yeastassay involves heterologously expressing GPR 41, GPR 42 or a variantpolypeptide in a modified yeast strain containing multiple reportergenes, typically FUS1-HIS3 and FUS1-lacZ, each linked to an endogenousMAPK cascade-based signal transduction pathway. This pathway is normallylinked to pheromone receptors, but can be coupled to foreign receptorsby replacement of the yeast G protein with yeast/mammalian G proteinchimeras. Strains may also contain further gene deletions, such asdeletions of SST2 and FAR1, to potentiate the assay. Ligand activationof the heterologous receptor can be monitored for example either as cellgrowth in the absence of histidine or with a suitable substrate such asbeta-galactosidase (lacZ).

[0078] Alternatively melanophore assays may be used to screen foractivators of GPR 41 or GPR 42. GPR 41, GPR 42 or a variant polypeptidecan be heterologously expressed in Xenopus laevis melanophores and theiractivation can be measured by either melanosome dispersion oraggregation. Basically, melanosome dispersion is promoted by activationof adenylate cyclase or phospholipase C, i.e. G_(s) and G_(q) mediatedsignalling respectively, whereas aggregation results from activation ofG_(i)-protein resulting in inhibition of adenylate cyclase. Hence,ligand activation of the heterologous receptor can be measured simply bymeasuring the change in light transmittance through the cells or byimaging the cell response.

[0079] Preferably, control experiments are carried out on cells which donot express GPR 41, GPR 42 or a variant polypeptide to establish whetherthe observed responses are the result of activation of the GPR 41, GPR42 or the variant polypeptide. Competitive assays may be carried out ona test substance in the presence of a known activator or antagonist ofGPR 41 or GPR 42.

[0080] In vitro assay systems to measure lipolysis include cell linesthat can be induced to differentiate into adipocytes such as3T3-L1(murine) and SAOS-2(human) cells (Imamura et al, J. Biol. Chem.274, 33691-33695, 1999; Diascro et al, J. Bone & Mineral Res. 13,96-106, 1998). Alternatively, primary adipocytes harvested from ananimal or human donor may be used.

[0081] Additional assays may thus be carried out in adipocytes. Forexample, the hydrolysis of triglycerides (TG) to non-esterified fattyacids (NEFA) and glycerol is performed by hormone-sensitive lipase(HSL). The activity of HSL is regulated by cAMP-dependent proteinkinases. Therefore, inhibition of cAMP generation by adenylate cyclasevia G_(i)-coupled receptors (e.g. GPR 41 or GPR 42 or a variant ofeither thereof) results in the reduction of NEFA and glycerol levelsgenerated by adipocytes. Chromogenic assays for both NEFA and glycerolare commercially available (Randox) and can be used to verify thatpre-treatment of adipocytes with an agonist for GPR 41 or GPR 42 resultsin a reduction in the levels of NEFA and glycerol derived fromadipocytes. In addition, assays can be performed to measure the cAMPcontent of adipocytes in the presence and absence of modulators for GPR41, GPR 42 or a variant thereof in order to correlate reduction in theproducts of lipolysis with the activation of a Gi-coupled receptor.

[0082] A standard method for identifying lipolysis inhibitors is asfollows. Adipocytes, for example approximately 100,000 in 0.5 ml, arepre-treated with an agent under test. The pre-treated adipocytes areincubated in the presence of adenosine deaminase, thereby to preventaccumulation of endogenous adenosine. Incubation can be carried out for30 minutes at 37° C. Cells are centrifuged and buffer withdrawn frombelow the cell layer, heated such as at 70° C. for 10 minutes andglycerol can be assayed enzymatically. A suitable assay method isdescribed in McGowan et al, Clin. Chem. 29, 538-543, 1983).

[0083] Suitable test substances which can be tested in the above assaysinclude combinatorial libraries, defined chemical entities, peptide andpeptide mimetics, oligonucleotides and natural product libraries, suchas display (e.g. phage display libraries) and antibody products. In apreferred embodiment, the test substance is a nicotinic acid (Niacin).Assays may also be carried out using known ligands of other G-proteincoupled receptors to identify ligands which act as agonists at GPR 41 orGPR 42.

[0084] Test substances may be used in an initial screen of, for example,10 substances per reaction, and the substances of these batches whichshow inhibition or activation tested individually. Test substances maybe used at a concentration of from 1 nM to 1000 μM, preferably from 1 μMto 100 μM, more preferably from 1 μM to 10 μM.

[0085] Agents which modulate GPR 41 or GPR 42 activity and which havebeen identified by assays in accordance with the invention can be usedin the treatment or prophylaxis of lipid disorders which are responsiveto regulation of GPR 41 or GPR 42 receptor activity. Agents whichactivate GPR 41 or GPR 42 receptor activity and/or which have beenidentified as inhibitors of lipolysis are preferred. In particular, suchagents may be used in the treatment of dyslipidaemia and conditionsassociated with dyslipidaemia such as atherosclerosis, obesity,thrombosis or coronary artery disease, angina, chronic renal failure,peripheral vascular disease, stroke, type II diabetes, and metabolicsyndrome (syndrome X).

[0086] The agents may be formulated with a pharmaceutically acceptablecarrier and/or excipient as is routine in the pharmaceutical art. Seefor example Remington's Pharmaceutical Sciences, Mack PublishingCompany, Eastern Pennsylvania 17^(th) Ed. 1985. The carrier or excipientmay be an isotonic saline solution but will depend more generally uponthe particular agent concerned and the route by which the agent is to beadministered.

[0087] The agents may be administered by enteral or parenteral routessuch as via oral, buccal, anal, pulmonary, intravenous, intra-arterial,intramuscular, intraperitoneal, topical or other appropriateadministration routes. A therapeutically effective amount of a modulatoris administered to a patient. The dose of a modulator may be determinedaccording to various parameters and especially according to thesubstance used; the age, weight and condition of the patient to betreated; the route of administration; and the required regimen. Aphysician will be able to determine the required route of administrationand dosage for any particular patient. A typical daily dose is fromabout 0.1 to 50 mg per kg of body weight, according to the activity ofthe specific modulator, the age, weight and conditions of the subject tobe treated, the type and severity of the degeneration and the frequencyand route of administration. Preferably, daily dosage levels are from 5mg to 2 g.

[0088] Alternatively agents which up-regulate GPR 41 or 42 expression ornucleic acid encoding GPR 41, GPR 42 or a variant polypeptide may beadministered to the mammal. Nucleic acid, such as RNA or DNA, preferablyDNA, is provided in the form of a vector, which may be expressed in thecells of a human or other mammal under treatment. Preferably suchup-regulation or expression following nucleic acid administration willenhance GPR 41 or GPR 42 activity.

[0089] Nucleic acid encoding the GPR 41, GPR 42 or variant polypeptidemay be administered to a human or other mammal by any availabletechnique. For example, the nucleic acid may be introduced by injection,preferably intradermally, subcutaneously or intramuscularly.Alternatively, the nucleic acid may be delivered directly across theskin using a nucleic acid delivery device such as particle-mediated genedelivery. The nucleic acid may be administered topically to the skin, orto the mucosal surfaces for example by intranasal, oral, intravaginal,intrarectal administration.

[0090] Uptake of nucleic acid constructs may be enhanced by severalknown transfection techniques, for example those including the use oftransfection agents. Examples of these agents includes cationic agents,for example, calcium phosphate and DEAE-Dextran and lipofectants, forexample, lipofectam and transfectam. The dosage of the nucleic acid tobe administered can be altered. Typically the nucleic acid isadministered in the range of 1 pg to 1 mg, preferably to 1 pg to 10 μgnucleic acid for particle mediated gene delivery and 10 μg to 1 mg forother routes.

[0091] Polynucleotides encoding GPR 41, GPR 42 or a variant polypeptidecan also be used to identify mutation(s) in GPR 41 or GPR 42 genes whichmay be implicated in human disorders. Identification of such mutation(s)may be used to assist in diagnosis of dyslipidaema and conditionsassociated with dyslipidaemia such as, atherosclerosis, obesity,thrombosis, angina, chronic renal failure, peripheral vascular disease,stroke, type II diabetes, and metabolic syndrome (syndrome X) or otherdisorders or susceptibility to such disorders and in assessing thephysiology of such disorders.

[0092] Antibodies (either polyclonal or preferably monoclonalantibodies, chimeric, single chain, Fab fragments) which are specificfor the GPR 41 or GPR 42 polypeptide or a variant thereof can begenerated. Such antibodies may for example be useful in purification,isolation or screening methods involving immunoprecipitation techniquesand may be used as tools to elucidate further the function of GPR 41,GPR 42 or a variant thereof, or indeed as therapeutic agents in theirown right. Such antibodies may be used to block ligand binding to thereceptor. A variety of protocols for competitive binding orimmunoradiometric assays to determine the specific binding capability ofan antibody are well known in the art (see for example Maddox et al, J.Exp. Med. 158, 1211 etseq, 1993).

[0093] The following Examples illustrate the invention.

EXAMPLE 1

[0094] Taqman™ distribution analysis of GPR 41 and GPR 42 was carriedout to study expression of GPR 41 and GPR 42 in normal human tissues.The results for GPR 41 are shown in FIG. 1; those for GPR 42 are shownin FIG. 12. These demonstrate that expression of both GPR 41 and GPR 42is essentially restricted to adipose tissue.

EXAMPLE 2

[0095] Mammalian cells, such as HEK293, CHO and COS7 cells,over-expressing GPR 41, GPR 42 or a variant polypeptide are generatedfor use in the assay. 96 and 384 well plate, high throughput screens(HTS) are employed using fluorescence based calcium indicator molecules,including but not limited to dyes such as Fura-2, Fura-Red, Fluo 3 andFluo 4 (Molecular Probes). Secondary screening involves the sametechnology. Tertiary screens involve the study of modulators in rat,mouse and guinea-pig models of disease relevant to the target.

[0096] A screening assay may be conducted as follows. Mammalian cellsstably over-expressing the relevant polypeptide are cultured in blackwall, clear bottom, tissue culture-coated 96 or 384 well plates with avolume of 100 μl cell culture medium in each well 3 days before use in aFLIPR (Fluorescence Imaging Plate Reader—Molecular Devices). Cells areincubated with 4 μM FLUO-3AM at 30° C. in 5% CO₂ for 90 mins and arethen washed once in Tyrodes buffer containing 3 mM probenecid. Basalfluorescence is determined prior to addition of agents to be tested. TheGPR 41, GPR 42 or variant polypeptide is activated upon the addition ofa known agonist. Activation results in an increase in intracellularcalcium which can be measured directly in the FLIPR. For antagoniststudies, test agents are preincubated with the cells for 4 minutesfollowing dye loading and washing and fluorescence is measured for 4minutes. Agonists are then added and cell fluorescence measured for afurther 1 minute.

[0097]Xenopus oocyte expression may be determined as follows. Adultfemale Xenopus laevis (Blades Biologicals) are anaesthetised using 0.2%tricaine (3-aminobenzoic acid ethyl ester), killed and the ovariesrapidly removed. Oocytes are then de-folliculated by collagenasedigestion (Sigma type I, 1.5 mg ml⁻¹) in divalent cation-free OR2solution (82.5 mM NaCl, 2.5 mM KCl, 1.2 mM NaH₂PO₄, 5 mM HEPES; pH 7.5at 25° C.). Single stage V and VI oocytes are transferred to ND96solution (96 mM NaCl, 2 mM KCl, 1 mM MgCl₂, 5 mM HEPES, 2.5 mM sodiumpyruvate; pH 7.5 at 25° C.) which contains 50 μg ml⁻¹ gentamycin and arestored at 18° C.

[0098] The GPR 41 or GPR 42 receptor (in pcDNA₃, Invitrogen) islinearised and transcribed to RNA using T7 (Promega Wizard kit).m′G(5′)pp(5′)GTP capped cRNA is injected into oocytes (20-50 ng peroocyte) and whole-cell currents are recorded using two-microelectrodevoltage-clamp (Geneclamp amplifier, Axon instruments Inc.) 3 to 7 dayspost-RNA injection. Microelectrodes have a resistance of 0.5 to 2 MΩwhen filled with 3M KCl.

EXAMPLE 3

[0099] Transient transfection of the cDNA for human GPR41 together withthat of the G_(i) family G protein G_(o1α) into HEK293T cells led toacetate-mediated stimulation of [³⁵S]GTPγS binding on membranes fromharvested cells (FIG. 2). Such responses were not observed in cellstransfected with G_(o1α) alone. Further functional analysis demonstratedthat expression of GPR41 permitted responses of differing magnitude andpotency to a variety unsaturated carboxylic acids ranging from 1 carbonto 5 carbons in chain length (C1-C5) (FIG. 3), with propionate (C3)being the most potent and efficacious. In addition, the naturallyoccurring ketone body, 3-hydroxybutyrate, was also found to elevate[³⁵S]GTPγS binding on membranes from GPR41-expressing cells (FIG. 4).These data suggest that activation of GPR 41 by short chain carboxylicacids promotes activation Gi mediated signalling pathways, which inadipose tissue may lead to inhibition of lipolysis.

EXAMPLE 4

[0100] The rat orthologue of human GPR41 was identified as follows. Themouse orthologue of human GPR41/42 was identified by searching publicdomain databases with the peptide sequence for human GPR41. The GenBankentry Accession No. AC079472 contains the high throughput draft sequencefor mus musculus clone RP23-123D23. An open reading frame of 960 bp wasidentified (between residues 53091-52135) that was 72% homologous to thehuman sequence at both the DNA and protein level. This open readingframe was shorter (957 bp/319 amino acids vs 1038 bp/346 amino acids)than the corresponding human sequence. PCR primers were designed thatstarted immediately upstream of the putative start codon (NF4155′-CATTAGCATCTGTGATG-3′) (SEQ ID NO: 7) and that finished at theputative stop codon (NF416 5′-CTAGCTCGGACACTCCTTGG-3′) (SEQ ID NO: 8).Primers NF415 and NF416 were used to amplify the corresponding sectionof rat genomic DNA. This region was amplified using Pfu DNA polymeraseunder conditions recommended by the manufacturer (Stratagene) at anannealing temperature of 50° C. The fragment was cloned into the vectorPCR-Script (Stratagene) and sequenced. The DNA sequence was 91%homologous to murine GPR41 and translated amino acid sequence 92%homologous.

[0101] Since primer NF416 encoded amino acids based on the murinesequence another section of rat DNA was amplified using primers NF417(corresponding to the murine sequence 52 bp downstream of the stop codon5′-GCCATAGCACTGAGCCAATG-3′) (SEQ ID NO: 9) and NF412(5′-TTGTAGCCACGTTGCTCATC-3+ corresponding to residues 668-687 of the ratcoding sequence) (SEQ ID NO: 10). These primers were used to amplify afragment approximately 340 bp long extending beyond the putative stopcodon into the 3′ untranslated region. This region was amplified usingTaq DNA polymerase under conditions recommended by the manufacturer(Sigma) at an annealing temperature of 44° C. The fragment was clonedinto the vector pBluescript KS (Stratagene) and sequenced. Analysis ofthis clone gave the 3′ sequence of rat GPR41 up to and beyond the stopcodon. The DNA sequence of rat GPR41 is 70% homologous to human GPR41and translated amino acid sequence is 71% homologous.

[0102] Primers NF419 (5′-TAGGATCCATGGACACAAGCTTCTTCC-3′) (SEQ ID NO: 11)and NF420 (5′-TACTCGAGCTAGCTCGGACATTCCTTGGA-3′) (SEQ ID NO: 12) weredesigned to amplify the entire coding sequence of rat GPR41 and to addflanking 5′ BamHI and 3′ XhoI restriction sites. This region wasamplified using Pfu DNA polymerase under conditions recommended by themanufacturer (Stratgene) at an annealing temperature of 54° C. Thefragment was cloned into the vector PCR-Script (Stratagene) andsequenced. Sequence information for GPR 41 is provided in SEQ ID NO 5(nucleotide and amino acid) and in SEQ ID NO: 6 (amino acid). The insertwas removed as a BamHI/Xhol fragment and subcloned into the mammalianexpression vector pCDNA3 (invitrogen) and yeast expression vectorp426GPD.

EXAMPLE 5

[0103] It was shown that expression of rat GPR41 (rGPR41) in HEK293Tcells together with G_(o1α) also elicited carboxylic acid-mediatedstimulation of [³⁵S]GTPγS binding (FIG. 5). A similar rank order ofpotency for C1-C5 carboxylic acids was found between human and rGPR41suggesting similar pharmacological profiles for the two specieshomologues.

EXAMPLE 6

[0104] It was found that rGPR41 could be expressed in the yeastSaccharomyces cerevisiae and successfully coupled to the pheromoneresponse pathway. Significant absorbance at 570 nm, corresponding toinduction of FUS1-lacZ and FUS1-HIS3 reporter genes, was detected forMMY11 cells containing p426GPD-rGPR41 in combination withpRS314-Gpa1/G_(αo). These cells express rGPR41 in combination with a Gαsubunit identical to Gpa1 but in which the 5 C-terminal amino acids arereplaced with the 5 C-terminal amino acids of the mammalian Gα subunit,G_(αo). This response is dependant on the presence of the agonist ligandpropionate since no induction is detected in the absence of propionate(FIG. 6). Control cells transformed with the vector p426GPD incombination with pRS314-Gpa1/G_(αo) and therefore lacking rGPR41 did notrespond to propionate. Reporter gene activity was also detected in thepresence of the Gpa1/G_(α13) transplant, but this was observed even inthe absence of propionate and hence does not correspond to receptorcoupling but instead to the elevated basal reporter gene expressionobserved previously with the Gpa1/G_(α13) protein (Brown, 1999). Of theother Gα subunits tested, the pRS314-Gpa1/G_(α12) andpRS314-Gpa1/G_(α13) transplants supported weak rGPR41 couplingdetectable after 48 hrs incubation (data not shown), and with no otherGα subunit could ligand responses of rGPR41 be detected. The activationof the yeast signal transduction pathway in response to agonist, and thespecificity of activation of a particular Gα subunit, confirm that therGPR41 nucleotide sequence encodes a functional G-protein coupledreceptor which can be activated by propionate. Furthermore, thespecificity for Gpa1/G_(αo) combined with the weaker activation ofGpa1/G_(αi2) and pRS314-Gpa1/G_(αi3), suggests that under physiologicalcircumstances in mammalian cells rGPR41 is coupled to the Gi-family ofG-proteins.

EXAMPLE 7

[0105] A study was carried out of the function of rGPR41 followingexpression in S. cerevisiae MMY22, which is a derivative of S.cerevisiae MMY11 containing a copy of the gene encoding the Gpa1/G_(αo)transplant chimera described above (in which the 5 C-terminal aminoacids of Gpa1 are replaced with the 5 C-terminal amino acids of G_(αo))integrated into the genome. To do this, a cassette comprising the GPA1promoter, nucleotide sequence encoding Gpa1/G_(αo), and the terminatorwas sub-cloned from pRS314-Gpa1/G_(αo) into the yeast integratingplasmid pRS304 (Sikorski and Hieter, 1989). The resulting plasmidpRS304-Gpa1/G_(αo) was transformed into MMY11 and integrated into thetrp1 locus creating MMY22.

[0106] The rGPR41 expression construct described above, p462GPD-rGPR41was introduced into MMY22 by transformation as described above. Fourseparate transformants were tested for reporter gene activation inresponse to propionate. Assays for FUS1-lacZ and FUS1-HIS3 inductionwere performed as described, except the β-galactosidase (lacZ) substratefluorescein di-β-D-galactopyranoside (FDG) was used in place of CPRG,and 3-aminotriazole concentration was 5 mM. Also, black-walled 96-wellmicrotitre plates were used, and fluorescence resulting from degradationof FDG to fluorescein due to β-galactosidase was determined using aSpectrofluor microtitre plate reader (Tecan)(excitation wavelength: 485nm; emmision wavelength: 535 nm).

[0107] Significant fluorescence corresponding to activation of thepheromone response pathway by rGPR41 was detected. The extent of thisresponse was dependent on the concentration of propionate, and onlysmall basal levels of fluorescence were detected in the absence ofpropionate (FIG. 7). This result confirms the ability of the Gpa1/G_(αo)transplant chimera to support activation of the pheromone responsepathway by rGPR41 in response to its agonist ligand. The magnitude ofresponses to the highest propionate concentration varied amongsttransformants. This is likely to be due to small variations incopy-number of the p462GPD-rGPR41 plasmid, which are expected to vary(approximately 20 to 40 plasmid copies per cell) due to the 2 μ geneticelement on this plasmid. This is likely to cause small variations in thelevel of rGPR41 protein.

EXAMPLE 8

[0108] A series of carboxylic acids and other compounds related topropionate were tested for the ability to activate rGPR41. Experimentswere performed as described above, using yeast cells of strain MMY22transformed with rGPR41 expression plasmid p462GPD-rGPR41, except thatthe agonist propionate was replaced with one of a series of othercompounds, which are listed in table 2 below. TABLE 2 Compounds testedfor ability to activate GPR 41 Agonism @ rGPR41 Anion Form testedApprox. EC50 (M) 4-pentenoate 3.0E−05 acetamide very weak active AcetateSodium acetate 1.0E−03 Acetoacetate Lithium Acetoacetate weak activeAcetone free Inactive Alanine free Inactive alpha-hydroxybutyrate Sodiumalpha- Inactive hydroxybutyrate Ascorbate Sodium ascorbate Inactivebenzoate Sodium benzoate Toxic Caproic C6 acid Potassium salt from acid3.0E−05 Caprylic C8 acid Potassium salt from acid weak active CitratePotassium salt from acid Inactive D-b-Hydroxybutyrate Sodium salt1.0E−02 Decanoic C10 acid Potassium salt from acid inactive/toxicDL-b-Hydroxybutyrate Sodium beta- 1.0E−02 hydroxybutyrate FormateAmmonium formate Weak active Fumarate Inactive GABA Inactiveγ-hydroxybutyrate Sodium gamma- 1.0E−02 hydroxybutyrate Glutamate Sodiumglutamate Inactive Glycine free Inactive glycolate weak active Glyoxalfree Toxic Glyoxylic acid sodium glyoxylate 5.0E−02 Heptanoic C7 acidPotassium salt from acid 1.0E−04 Isobutyrate 3.0E−05 lactate sodiumlactate Inactive lactate Potassium salt from acid Inactive Lauric C12acid Potassium salt from acid inactive/toxic L-b-Hydroxybutyrate Sodiumsalt 1.0E−02 Linoleic acid Toxic/Inactive maleate Potassium salt fromacid Inactive malonate Sodium malonate Inactive methyl-2-imidazolineToxic/inactive methylphosphonate Inactive methylsulphonate Sodiummethylsulphonate Inactive n-Butyrate Potassium salt from acid 5.0E−05nicotinic acid Inactive NMDA Inactive Nonanoic C9 acid Potassium saltfrom acid inactive/toxic oleate Sodium oleate in 1% BSA Inactive (KOHadded by mistake) Oxalate Sodium oxalate 3.0E−03 Oxaloacetate Potassiumsalt from acid Inactive palmitate Sodium palmitate Inactive Palmitoleicacid Toxic/inactive Pentanoate (n-valerate) Potassium salt from acid3.0E−06 pivalic acid 5.0E−04 Propionate Sodium propionate 5.0E−06pyruvate Inactive stearate Sodium stearate Inactive Succinate disodiumsuccinate Inactive Tartrate Sodium tartrate Inactive tiglic acidInactive Trichloroacetate Potassium salt from acid ToxicTrifluoroacetate Na TFA weak active Undecanoic C11 acid Potassium saltfrom acid inactive/toxic

[0109] The compounds were generally organic anions, which wereintroduced into the assay as sodium or potassium salt solutions bufferedto pH 7.0. The extent of GPR 41 activation due to the compound tested isalso shown in Table 2. Where significant activity was detected, therelative activity is given as approximate EC50 value, where propionategave EC50 value of 5 μM. The most active compounds tested wereunsaturated, straight or branched chain carboxylic acids (short-chainfatty acids). Concentration-response curves for the series ofunsaturated, straight-chain carboxylic acids containing from 1 to 12carbon atoms is shown in FIG. 8. Pentanoate (n-valerate), the carboxylicacid containing five carbon atoms, is similarly or slightly more activethan propionate. The order of agonist potency of this series of ligandsis: C1<C2<C3=C5>C6>C7>C8, also C3>C4<C5, and C9, C10, C11 and C12 areinactive, where C1=formate, C2=acetate, C3=propionate, C4=butyrate,C5=pentanoate, C6=caproate, C7=heptanoate, C8=caprylate, C9=nonanoate,C10=decanoate, C11=undecanoate, and C12=dodecanoate. This experimentdemonstrates that positions of hydrogen atoms within a canonicalstraight chain, unsaturated carboxylic acid agonist (such as propionate,butyrate, or pentanoate) may be substituted for hydroxyl groups orfluorine atoms without abolishing activity, though not larger halogenssuch as chlorine. Substitution with methyl groups giving branched chains(as in isobutyric acid and pivalic acid) or carbon-carbon double bondsgiving unsaturated chains (as in 4-pentenoate) also may be toleratedwithout abolishing activity. The carboxylic acid group appears to berequired for activity. A series of naturally occurring compoundscontaining multiple carboxylic acids (succinate, fumarate, citrate, etc)were either inactive or had very weak activities.

EXAMPLE 9

[0110] A study was made of the agonism of rGPR41 by thenaturally-occurring ketone body compound, 3-hydroxybutyrate. Experimentswere performed as described above, using yeast cells of strain MMY22transformed with rGPR41 expression plasmid p462GPD-rGPR41, except thatthe substrate CPRG was used. Also, the agonist propionate was replacedwith 3-hydroxybutyrate (β-hydroxybutyrate), either as a racemic mix ofboth stereoisomers, or as purified enantiomers D-3-hydroxybutyrate, orL-3-hydroxybutyrate. 3-hydroxybutyrate acted as an agonist at rGPR41with a potency similar or slightly less than acetate (FIG. 9). Thelowest concentrations of β-hydroxybutyrate which caused detectableactivation of rGPR41 were in the range 0.1 mM to 1 mM. The stereoisomershad broadly similar activities: D-3-hydroxybutyrate was similarly activeto the racemic mix DL-3-hydroxybutyrate, and possibly slightly moreactive at the highest concentration tested than L-3-hydroxybutyrate.

EXAMPLE 10

[0111] Carboxylic acids were tested for the ability to alterG_(i)-mediated signalling mechanisms in adipocytes. Application of C1-C4to freshly isolated primary adipocytes from rat eididymal fat pads ledto significant inhibition of isoprenaline-stimulated lipolysis in aconcentration-dependent manner (FIG. 10). We also demonstrated that overa similar concentration range acetate (C2) also caused a reduction ofisoprenaline-amplified cAMP levels (FIG. 11). Isoprenaline stimulatesadenylate cyclase activity and subsequently the process of lipolysisfollowing activation of β-adrenoceptors and G,a in adipocytes. Hence,inhibition of these activities is normally via receptor-mediatedstimulation of G_(i) G protein signalling. These data thereforedemonstrate that carboxylic acids which are ligands at GPR41, a receptorwhich is highly expressed in adipose tissue, can regulate G_(i) Gprotein signalling pathways mediated possibly via direct activation ofGPR41.

[0112] Of the various compounds acting as GPR 41 agonists identifiedfrom screening the ketone body compound 3-hydroxyutyrate is ofparticular interest as a GPR 41 agonist, as it is known to occurphysiologically in blood, and may have effects in adipocyte lipolysis.The presence of GPR 41, a receptor responsive to 3-hydroxybutyrate onadipocytes, suggests that GPR 41 may mediate 3-hydroxybutyrate inducedinhibition of lipolysis and/or increased adipocyte sensitivity toinsulin. A role for GPR 41 in regulating lipolysis is further supportedby demonstration that this receptor is Gi coupled and is thereforeexpected to inhibit adenylate cyclase on activation, since the lipaseresponsible for lipolysis is regulated by cAMP levels. Also inheterologous assays described above, the sensitivity of GPR 41 to3-hydroxybutyrate is in the physiological concentration range, andfurther more the threshold of GPR 41 activation occurs at very close tolevels which would cause acidosis (approximately 1 mM see FIG. 9).

[0113] Methods

[0114] Mammalian Cell Culture and Transfections

[0115] HEK293T cells (HEK293 cells stably expressing the SV40 largeT-antigen) were maintained in DMEM containing 10% (v/v) foetal calfserum and 2 mM glutamine. Cells were seeded in 60 mm culture dishes andgrown to 60-80% confluency (18-24 h) prior to transfection with pCDNA3containing the relevant DNA species using Lipofectamine reagent. Fortransfection, 3 μg of DNA was mixed with 10 μl of Lipofectamine in 0.2ml of Opti-MEM (Life Technologies Inc.) and was incubated at roomtemperature for 30 min prior to the addition of 1.6 ml of Opti-MEM.Cells were exposed to the Lipofectamine/DNA mixture for 5 h and 2 ml of20% (v/v) newborn calf serum in DMEM was then added. Cells wereharvested 48-72 h after transfection.

[0116] Preparation of Membranes

[0117] Plasma membrane-containing P2 particulate fractions were preparedfrom cell pastes frozen at −80° C. after harvest. All procedures werecarried out at 4° C. Cell pellets were resuspended in 1 ml of 10 mMTris-HCl and 0.1 mM EDTA, pH 7.5 (buffer A) and by homogenisation for 20s with a polytron homogeniser followed by passage (5 times) through a25-guage needle. Cell lysates were centrifuged at 1,000 g for 10 min ina microcentrifuge to pellet the nuclei and unbroken cells and P2particulate fractions were recovered by microcentrifugation at 16,000 gfor 30 min. P2 particulate fractions were resuspended in buffer A andstored at −80° C. until required. Protein concentrations were determinedusing the bicinchoninic acid (BCA) procedure (4) using BSA as astandard.

[0118] High Affinity [³⁵S]GTPγS Binding

[0119] Assays were performed in 96-well format using a method modifiedfrom Wieland and Jakobs, 1994. Membranes (10 μg per point) were dilutedto 0.083 mg/ml in assay buffer (20 mM HEPES, 100 mM NaCl, 10 mM MgCl₂,pH7.4) supplemented with saponin (10 mg/l) and pre-incubated with 40 μMGDP. Various concentrations of nicotinic acid were added, followed by[³⁵S]GTPγS (1170 Ci/mmol, Amersham) at 0.3 nM (total vol. of 100 μl) andbinding was allowed to proceed at room temperature for 30 min.Non-specific binding was determined by the inclusion of 0.6 mM GTP.Wheatgerm agglutinin SPA beads (Amersham) (0.5 mg) in 25 μl assay bufferwere added and the whole was incubated at room temperature for 30 minwith agitation. Plates were centrifuged at 1500 g for 5 min and bound[³⁵S]GTPγS was determined by scintillation counting on a Wallac 1450microbeta Trilux scintillation counter.

[0120] Construction of p426GPD-rGPR41 for Expression of rGPR41 in YeastCells

[0121] Nucleotide sequence encoding rGPR41 flanked by restriction enzymesites BamHI and XhoI was cloned into the yeast expression vector p426GPD(Mumberg, 1995). The orientation of insertion was such that, whenintroduced into yeast cells, transcription from the GPD promotercontained within the p426GPD-rGPR41 plasmid resulted in production ofmRNA encoding rGPR41 protein. The GPD promoter sequence in p426GPD is acopy of the chromosomal sequence upstream of the highly expressed yeastgene, TDH1. Hence, yeast cells containing p426GPD-rGPR41 should producerGPR41 protein to high levels.

[0122] Transformation of Yeast Strain MMY11 with Constructp426GPD-rGPR41 in Combination with Gα Subunit Expression Constructs

[0123] The yeast strain MMY11 has been described previously (Olesnickyet al, 1999). It contains a series of genetic modifications to enablecoupling of heterologously expressed receptors to the expression of tworeporter genes, via the endogenous yeast pheromone response signaltransduction pathway. Importantly, the gene encoding the endogenousyeast pheromone receptor, STE2, has been deleted from MMY1 1 such thatcells of strain MMY11 containing p426GPD-rGPR41 will express rGPR41protein in place of Ste2 receptor protein. Furthermore, the geneencoding the G-protein α-subunit involved in the pheromone response,GPA1, has been deleted from MMY11. To enable receptor coupling in strainMMY11, plasmid constructs encoding either wild-type GPA1 of modifiedversions of GPA1 are introduced into MMY11 and are expressed in place ofendogenous yeast GPA1. The series of plasmids encoding modified versionsof GPA1 has been described previously (Brown et al., 1999) and is thesubject of patent (application number PCT.GB98.02759). Generally, themodifications made to Gpa1 facilitate coupling of heterologouslyexpressed receptors to the yeast pheromone response pathway. Yeaststrain MMY11 was transformed with pairs of plasmids, the first beingp426GPD-rGPR41 and the second being one of the pRS314-based Gαexpression constructs from table 3 below. Yeast transformations wereperformed according to the routine methods (Gietz et al., 1992). TABLE 3pRS314-based Gα expression constructs used in this experiment:Modification to Gpal C- Nature of Gα Gα expression construct terminussubunit pRS314-Gpal None Wild-type Gpal pRS314-Gpal/G_(αq)  5 aminoacids replaced Chimera (transplant) pRS314-Gpal/G_(αs)  5 amino acidsreplaced Chimera (transplant) pRS314-Gpal/G_(αo)  5 amino acids replacedChimera (transplant) pRS314-Gpal/G_(αi2)  5 amino acids replaced Chimera(transplant) pRS314-Gpal/G_(αi3)  5 amino acids replaced Chimera(transplant) pRS314-Gpal/G_(αz)  5 amino acids replaced Chimera(transplant) pRS314-Gpal/G_(α12)  5 amino acids replaced Chimera(transplant) pRS314-Gpal/G_(α13)  5 amino acids replaced Chimera(transplant) pRS314-Gpal/G_(α14)  5 amino acids replaced Chimera(transplant) pRS314-Gpal/G_(α16)  5 amino acids replaced Chimera(transplant) pRS314-Gpal-G_(αo) 142 amino acids replaced ChimerapRS314-Gpal-G_(αi1) 142 amino acids replaced Chimera pRS314-Gpal-G_(αi2)142 amino acids replaced Chimera pRS314-Gpal/G_(αi3) 142 amino acidsreplaced Chimera

[0124] Assay for Induction of Reporter Genes FUS1-lacZ and FUS1-HIS3 inResponse to GPR41 Ligands

[0125] In vivo assays of reporter gene induction were carried out bysuspending MMY11 cells transformed as described above to a density of0.02 OD₆₀₀/ml in 200 μl SC-glucose (2%) medium lacking tryptophan,uracil and histidine. This medium was supplemented with 10 mM3-aminotriazole and the β-galactosidase (lacZ) substratechlorophenolred-β-D-galactopyranoside (CPRG; Boehringer Mannheim) to aconcentration of 0.1 mg/ml. Additionally the medium was supplementedwith various concentrations of the agonist ligand, propionate (sodiumpropionate, pH 7.0). To visualise the yellow to red colour changereaction occurring on degradation of CPRG due to β-galactosidase, themedium was buffered to pH 7 with 0.1 M sodium phosphate. The assay wasconducted in flat-bottomed sterile 96-well microtitre plates. Plateswere incubated for 24 hours at 30° C. without agitation, and absorbanceat 570 nm was determined using a Spectrofluor microtitre plate reader(Tecan).

[0126] Preparation of Rat Primary Adipocytes

[0127] 70 ml of the “collection buffer” is prepared freshly each day.This buffer consists of 2.8 g of BSA (Sigma: A 7030) dissolved in 70 mlof DMEM (HEPES modification, Sigma: D6171), to aid the dissolution ofthe BSA the media is incubated at 37° C. Approximately 30 ml of thebuffer were then transferred to a 70 ml Sterilin pot for the collectionof rat epididymal fat pads. The remainder of the buffer is used toprepare the collagenase solution, which is freshly prepared for eachexperiment. Collagenase Type II (25 mg; Sigma: C 6885) is dissolved into 25 ml of the “collection buffer” and supplemented with 80 μl of a 1MCaCl₂ solution (to give a final Ca²⁺ concentration of 5 mM) and 25 μl ofadenosine deaminase (Sigma: A 1030; final concentration=10 μg/ml).

[0128] The freshly removed fat pads are then individually weighed andthen cut in to small pieces and each fat pad are added to a 50 mlconical flask containing 12.5 ml of the 1 mg/ml collagenase solution. Nomore than 6 grams, wet weight, of adipose tissue is added to each 12.5ml volume of collagenase solution. The adipose tissue is then incubatedfor 60-75 minutes at 37° C. whilst being mixed at 150 cycles per minute.At the end of the incubation period the adipose tissue is filteredthrough a 100 μm mesh (Falcon) in to a 50 ml Falcon tube. In order tofacilitate the passage of the adipocytes through the filter they areflushed with Krebs buffer which has been supplemented with BSA (1%) andADA (10 μg/ml).

[0129] The adipocytes are then centrifuged (500 rpm, 1 minute) to allowthe adipocytes to float to the surface. The infranatant is removed, andthe volume made up to 35 ml by the addition of fresh Krebs Buffer. Theadipocytes were again centrifuged (500 rpm, 1 minute) and theinfranatant removed. This washing step is repeated and the residualadipocytes transferred to a 5ml Sterilin tube and kept at 37° C. priorto use.

[0130] Adipocyte Lipolysis Assays

[0131] The lipolysis assay was performed on a 24-well plate, in a volumeof 1 ml. 800 μl of Krebs buffer was added in to each well. Testcompounds or their vehicle was added as 100-fold concentrated stocks(i.e. 10 μl per well). Isoprenaline (100 μl of a 1 μM solution) wasadded to the relevant wells to give a final concentration of 100 nM.Finally the assay was started by the addition of 100 μl of the adipocytesuspension. The 24-well plate was then transferred to an incubator (37°C./5% CO₂) and left for 2 hours At the end of this incubation period, a25 μl samples were removed from each well and transferred to a 96-wellplate. The levels of glycerol were then determined by a commerciallyavailable assay (Randox).

[0132] In this test system several carboxylic acid derivatives weretested up to concentrations of 0.1 M. A concentration-related inhibitionof lipolysis was observed with these ligands. Inhibition of glycerol wasnot due to a direct effect in the assay since, in a no adipocytecontrol, none of the compounds tested effected the assay atconcentrations up to 1 M.

[0133] cAMP Quantification

[0134] A reaction mixture of 500 μl was used in a 1.5 ml eppendorf tube.The reaction was started by the addition of 100 μl of adipocytes to a400 μl volume of Krebs buffer containing isoprenaline and test compound.Following the addition of the adipocytes the reaction tube was incubatedat 37° C. for 10 minutes. The reaction was stopped with the addition of500 μl of a stop mixture containing methanol (1 part), chloroform (2parts) and 0.1N HCl (0.1 part). Following the addition of the stopmixture each tube was vortexed and then centifuged at 5000 rpm for 5minutes. 300 μl of the supernatent was removed and stored at −20° C. Thelevels of cAMP in each samples were determined using an ELISA kit fromR&D systems (DE0355).

1 12 1 1041 DNA Homo sapiens CDS (1)..(1041) 1 atg gat aca ggc ccc gaccag tcc tac ttc tcc ggc aat cac tgg ttc 48 Met Asp Thr Gly Pro Asp GlnSer Tyr Phe Ser Gly Asn His Trp Phe 1 5 10 15 gtc ttc tcg gtg tac cttctc act ttc ctg gtg ggg ctc ccc ctc aac 96 Val Phe Ser Val Tyr Leu LeuThr Phe Leu Val Gly Leu Pro Leu Asn 20 25 30 ctg ctg gcc ctg gtg gtc ttcgtg ggc aag ctg cag cgc cgc ccg gtg 144 Leu Leu Ala Leu Val Val Phe ValGly Lys Leu Gln Arg Arg Pro Val 35 40 45 gcc gtg gac gtg ctc ctg ctc aacctg acc gcc tcg gac ctg ctc ctg 192 Ala Val Asp Val Leu Leu Leu Asn LeuThr Ala Ser Asp Leu Leu Leu 50 55 60 ctg ctg ttc ctg cct ttc cgc atg gtggag gca gcc aat ggc atg cac 240 Leu Leu Phe Leu Pro Phe Arg Met Val GluAla Ala Asn Gly Met His 65 70 75 80 tgg ccc ctg ccc ttc atc ctc tgc ccactc tct gga ttc atc ttc ttc 288 Trp Pro Leu Pro Phe Ile Leu Cys Pro LeuSer Gly Phe Ile Phe Phe 85 90 95 acc acc atc tat ctc acc gcc ctc ttc ctggca gct gtg agc att gaa 336 Thr Thr Ile Tyr Leu Thr Ala Leu Phe Leu AlaAla Val Ser Ile Glu 100 105 110 cgc ttc ctg agt gtg gcc cac cca ctg tggtac aag acc cgg ccg agg 384 Arg Phe Leu Ser Val Ala His Pro Leu Trp TyrLys Thr Arg Pro Arg 115 120 125 ctg ggg cag gca ggt ctg gtg agt gtg gcctgc tgg ctg ttg gcc tct 432 Leu Gly Gln Ala Gly Leu Val Ser Val Ala CysTrp Leu Leu Ala Ser 130 135 140 gct cac tgc agc gtg gtc tac gtc ata gaattc tca ggg gac atc tcc 480 Ala His Cys Ser Val Val Tyr Val Ile Glu PheSer Gly Asp Ile Ser 145 150 155 160 cac agc cag ggc acc aat ggg acc tgctac ctg gag ttc cgg aag gac 528 His Ser Gln Gly Thr Asn Gly Thr Cys TyrLeu Glu Phe Arg Lys Asp 165 170 175 cag cta gcc atc ctc ctg ccc gtg cggctg gag atg gct gtg gtc ctc 576 Gln Leu Ala Ile Leu Leu Pro Val Arg LeuGlu Met Ala Val Val Leu 180 185 190 ttt gtg gtc ccg ctg atc atc acc agctac tgc tac agc cgc ctg gtg 624 Phe Val Val Pro Leu Ile Ile Thr Ser TyrCys Tyr Ser Arg Leu Val 195 200 205 tgg atc ctc ggc aga ggg ggc agc caccgc cgg cag agg agg gtg gcg 672 Trp Ile Leu Gly Arg Gly Gly Ser His ArgArg Gln Arg Arg Val Ala 210 215 220 ggg ctg ttg gcg gcc acg ctg ctc aacttc ctt gtc tgc ttt ggg ccc 720 Gly Leu Leu Ala Ala Thr Leu Leu Asn PheLeu Val Cys Phe Gly Pro 225 230 235 240 tac aac gtg tcc cat gtc gtg ggctat atc tgc ggt gaa agc ccg gca 768 Tyr Asn Val Ser His Val Val Gly TyrIle Cys Gly Glu Ser Pro Ala 245 250 255 tgg agg atc tac gtg acg ctt ctcagc acc ctg aac tcc tgt gtc gac 816 Trp Arg Ile Tyr Val Thr Leu Leu SerThr Leu Asn Ser Cys Val Asp 260 265 270 ccc ttt gtc tac tac ttc tcc tcctcc ggg ttc caa gcc gac ttt cat 864 Pro Phe Val Tyr Tyr Phe Ser Ser SerGly Phe Gln Ala Asp Phe His 275 280 285 gag ctg ctg agg agg ttg tgt gggctc tgg ggc cag tgg cag cag gag 912 Glu Leu Leu Arg Arg Leu Cys Gly LeuTrp Gly Gln Trp Gln Gln Glu 290 295 300 agc agc atg gag ctg aag gag cagaag gga ggg gag gag cag aga gcg 960 Ser Ser Met Glu Leu Lys Glu Gln LysGly Gly Glu Glu Gln Arg Ala 305 310 315 320 gac cga cca gct gaa aga aagacc agt gaa cac tca cag ggc tgt gga 1008 Asp Arg Pro Ala Glu Arg Lys ThrSer Glu His Ser Gln Gly Cys Gly 325 330 335 act ggt ggc cag gtg gcc tgtgct gaa agc tag 1041 Thr Gly Gly Gln Val Ala Cys Ala Glu Ser 340 345 2346 PRT Homo sapiens TRANSMEM (18)..(41) TRANSMEM (52)..(73) TRANSMEM(88)..(111) TRANSMEM (132)..(153) TRANSMEM (188)..(212) TRANSMEM(229)..(250) TRANSMEM (259)..(278) 2 Met Asp Thr Gly Pro Asp Gln Ser TyrPhe Ser Gly Asn His Trp Phe 1 5 10 15 Val Phe Ser Val Tyr Leu Leu ThrPhe Leu Val Gly Leu Pro Leu Asn 20 25 30 Leu Leu Ala Leu Val Val Phe ValGly Lys Leu Gln Arg Arg Pro Val 35 40 45 Ala Val Asp Val Leu Leu Leu AsnLeu Thr Ala Ser Asp Leu Leu Leu 50 55 60 Leu Leu Phe Leu Pro Phe Arg MetVal Glu Ala Ala Asn Gly Met His 65 70 75 80 Trp Pro Leu Pro Phe Ile LeuCys Pro Leu Ser Gly Phe Ile Phe Phe 85 90 95 Thr Thr Ile Tyr Leu Thr AlaLeu Phe Leu Ala Ala Val Ser Ile Glu 100 105 110 Arg Phe Leu Ser Val AlaHis Pro Leu Trp Tyr Lys Thr Arg Pro Arg 115 120 125 Leu Gly Gln Ala GlyLeu Val Ser Val Ala Cys Trp Leu Leu Ala Ser 130 135 140 Ala His Cys SerVal Val Tyr Val Ile Glu Phe Ser Gly Asp Ile Ser 145 150 155 160 His SerGln Gly Thr Asn Gly Thr Cys Tyr Leu Glu Phe Arg Lys Asp 165 170 175 GlnLeu Ala Ile Leu Leu Pro Val Arg Leu Glu Met Ala Val Val Leu 180 185 190Phe Val Val Pro Leu Ile Ile Thr Ser Tyr Cys Tyr Ser Arg Leu Val 195 200205 Trp Ile Leu Gly Arg Gly Gly Ser His Arg Arg Gln Arg Arg Val Ala 210215 220 Gly Leu Leu Ala Ala Thr Leu Leu Asn Phe Leu Val Cys Phe Gly Pro225 230 235 240 Tyr Asn Val Ser His Val Val Gly Tyr Ile Cys Gly Glu SerPro Ala 245 250 255 Trp Arg Ile Tyr Val Thr Leu Leu Ser Thr Leu Asn SerCys Val Asp 260 265 270 Pro Phe Val Tyr Tyr Phe Ser Ser Ser Gly Phe GlnAla Asp Phe His 275 280 285 Glu Leu Leu Arg Arg Leu Cys Gly Leu Trp GlyGln Trp Gln Gln Glu 290 295 300 Ser Ser Met Glu Leu Lys Glu Gln Lys GlyGly Glu Glu Gln Arg Ala 305 310 315 320 Asp Arg Pro Ala Glu Arg Lys ThrSer Glu His Ser Gln Gly Cys Gly 325 330 335 Thr Gly Gly Gln Val Ala CysAla Glu Ser 340 345 3 1041 DNA Homo Sapiens CDS (1)..(1041) 3 atg gataca ggc ccc gac cag tcc tac ttc tcc ggc aat cac tgg ttc 48 Met Asp ThrGly Pro Asp Gln Ser Tyr Phe Ser Gly Asn His Trp Phe 1 5 10 15 gtc ttctcg gtg tac ctt ctc act ttc ctg gtg ggg ctc ccc ctc aac 96 Val Phe SerVal Tyr Leu Leu Thr Phe Leu Val Gly Leu Pro Leu Asn 20 25 30 ctg ctg gccctg gtg gtc ttc gtg ggc aag ctg cgg tgc cgc ccg gtg 144 Leu Leu Ala LeuVal Val Phe Val Gly Lys Leu Arg Cys Arg Pro Val 35 40 45 gcc gtg gac gtgctc ctg ctc aac ctg acc gcc tcg gac ctg ctc ctg 192 Ala Val Asp Val LeuLeu Leu Asn Leu Thr Ala Ser Asp Leu Leu Leu 50 55 60 ctg ctg ttc ctg cctttc cgc atg gtg gag gca gcc aat ggc atg cac 240 Leu Leu Phe Leu Pro PheArg Met Val Glu Ala Ala Asn Gly Met His 65 70 75 80 tgg ccc ctg ccc ttcatc ctc tgc cca ctc tct gga ttc atc ttc ttc 288 Trp Pro Leu Pro Phe IleLeu Cys Pro Leu Ser Gly Phe Ile Phe Phe 85 90 95 acc acc atc tat ctc accgcc ctc ttc ctg gca gct gtg agc att gaa 336 Thr Thr Ile Tyr Leu Thr AlaLeu Phe Leu Ala Ala Val Ser Ile Glu 100 105 110 cgc ttc ctg agt gtg gcccac cca ctg tgg tac aag acc cgg ccg agg 384 Arg Phe Leu Ser Val Ala HisPro Leu Trp Tyr Lys Thr Arg Pro Arg 115 120 125 ctg ggg cag gca ggt ctggtg agt gtg gcc tgc tgg ctg ttg gcc tct 432 Leu Gly Gln Ala Gly Leu ValSer Val Ala Cys Trp Leu Leu Ala Ser 130 135 140 gct cac tgc agc gtg gtctac gtc ata gaa ttc tca ggg gac atc tcc 480 Ala His Cys Ser Val Val TyrVal Ile Glu Phe Ser Gly Asp Ile Ser 145 150 155 160 cac agc cag ggc accaat ggg acc tgc tac ctg gag ttc tgg aag gac 528 His Ser Gln Gly Thr AsnGly Thr Cys Tyr Leu Glu Phe Trp Lys Asp 165 170 175 cag cta gcc atc ctcctg ccc gtg cgg ctg gag atg gct gtg gtc ctc 576 Gln Leu Ala Ile Leu LeuPro Val Arg Leu Glu Met Ala Val Val Leu 180 185 190 ttt gtg gtc ccg ctgatc atc acc agc tac tgc tac agc cgc ctg gtg 624 Phe Val Val Pro Leu IleIle Thr Ser Tyr Cys Tyr Ser Arg Leu Val 195 200 205 tgg atc ctc ggc agaggg ggc agc cac cgc cgg cag agg agg gtg gcg 672 Trp Ile Leu Gly Arg GlyGly Ser His Arg Arg Gln Arg Arg Val Ala 210 215 220 ggg ctg gtg gcg gccacg ctg ctc aac ttc ctt gtc tgc ttt ggg ccc 720 Gly Leu Val Ala Ala ThrLeu Leu Asn Phe Leu Val Cys Phe Gly Pro 225 230 235 240 tac aac gtg tcccat gtc gtg ggc tat atc tgc ggt gaa agc ccg gtg 768 Tyr Asn Val Ser HisVal Val Gly Tyr Ile Cys Gly Glu Ser Pro Val 245 250 255 tgg agg atc tacgtg acg ctt ctc agc acc ctg aac tcc tgt gtc gac 816 Trp Arg Ile Tyr ValThr Leu Leu Ser Thr Leu Asn Ser Cys Val Asp 260 265 270 ccc ttt gtc tactac ttc tcc tcc tcc ggg ttc caa gcc gac ttt cat 864 Pro Phe Val Tyr TyrPhe Ser Ser Ser Gly Phe Gln Ala Asp Phe His 275 280 285 gag ctg ctg aggagg ttg tgt ggg ctc tgg ggc cag tgg cag cag gag 912 Glu Leu Leu Arg ArgLeu Cys Gly Leu Trp Gly Gln Trp Gln Gln Glu 290 295 300 agc agc atg gagctg aag gag cag aag gga ggg gag gag cag aga gcg 960 Ser Ser Met Glu LeuLys Glu Gln Lys Gly Gly Glu Glu Gln Arg Ala 305 310 315 320 gac cga ccagct gaa aga aag acc agt gaa cac tca cag ggc tgt gga 1008 Asp Arg Pro AlaGlu Arg Lys Thr Ser Glu His Ser Gln Gly Cys Gly 325 330 335 act ggt ggccag gtg gcc tgt gct gaa aac tag 1041 Thr Gly Gly Gln Val Ala Cys Ala GluAsn 340 345 4 346 PRT Homo Sapiens 4 Met Asp Thr Gly Pro Asp Gln Ser TyrPhe Ser Gly Asn His Trp Phe 1 5 10 15 Val Phe Ser Val Tyr Leu Leu ThrPhe Leu Val Gly Leu Pro Leu Asn 20 25 30 Leu Leu Ala Leu Val Val Phe ValGly Lys Leu Arg Cys Arg Pro Val 35 40 45 Ala Val Asp Val Leu Leu Leu AsnLeu Thr Ala Ser Asp Leu Leu Leu 50 55 60 Leu Leu Phe Leu Pro Phe Arg MetVal Glu Ala Ala Asn Gly Met His 65 70 75 80 Trp Pro Leu Pro Phe Ile LeuCys Pro Leu Ser Gly Phe Ile Phe Phe 85 90 95 Thr Thr Ile Tyr Leu Thr AlaLeu Phe Leu Ala Ala Val Ser Ile Glu 100 105 110 Arg Phe Leu Ser Val AlaHis Pro Leu Trp Tyr Lys Thr Arg Pro Arg 115 120 125 Leu Gly Gln Ala GlyLeu Val Ser Val Ala Cys Trp Leu Leu Ala Ser 130 135 140 Ala His Cys SerVal Val Tyr Val Ile Glu Phe Ser Gly Asp Ile Ser 145 150 155 160 His SerGln Gly Thr Asn Gly Thr Cys Tyr Leu Glu Phe Trp Lys Asp 165 170 175 GlnLeu Ala Ile Leu Leu Pro Val Arg Leu Glu Met Ala Val Val Leu 180 185 190Phe Val Val Pro Leu Ile Ile Thr Ser Tyr Cys Tyr Ser Arg Leu Val 195 200205 Trp Ile Leu Gly Arg Gly Gly Ser His Arg Arg Gln Arg Arg Val Ala 210215 220 Gly Leu Val Ala Ala Thr Leu Leu Asn Phe Leu Val Cys Phe Gly Pro225 230 235 240 Tyr Asn Val Ser His Val Val Gly Tyr Ile Cys Gly Glu SerPro Val 245 250 255 Trp Arg Ile Tyr Val Thr Leu Leu Ser Thr Leu Asn SerCys Val Asp 260 265 270 Pro Phe Val Tyr Tyr Phe Ser Ser Ser Gly Phe GlnAla Asp Phe His 275 280 285 Glu Leu Leu Arg Arg Leu Cys Gly Leu Trp GlyGln Trp Gln Gln Glu 290 295 300 Ser Ser Met Glu Leu Lys Glu Gln Lys GlyGly Glu Glu Gln Arg Ala 305 310 315 320 Asp Arg Pro Ala Glu Arg Lys ThrSer Glu His Ser Gln Gly Cys Gly 325 330 335 Thr Gly Gly Gln Val Ala CysAla Glu Asn 340 345 5 960 DNA Rattus sp. CDS (1)..(960) 5 atg gac acaagc ttc ttt ccc ggc aac cac tgg ctt ttc ttt tca gtg 48 Met Asp Thr SerPhe Phe Pro Gly Asn His Trp Leu Phe Phe Ser Val 1 5 10 15 gat ctg ttggtg ttc ctc gtg gga cta ccc ctc aac gtg atg gcc ctg 96 Asp Leu Leu ValPhe Leu Val Gly Leu Pro Leu Asn Val Met Ala Leu 20 25 30 gtg gtc ttc gtgaac aag ctg cgt cgc cgc ccg gtg gcc gtg gac tta 144 Val Val Phe Val AsnLys Leu Arg Arg Arg Pro Val Ala Val Asp Leu 35 40 45 ctt ttg ctt aac ctgacc att tcg gac ctg ctt ctg ctc ctc ttc ctg 192 Leu Leu Leu Asn Leu ThrIle Ser Asp Leu Leu Leu Leu Leu Phe Leu 50 55 60 cca ttc cgt ata gtg gaggcg gcc tgt ggc atg aaa tgg att ctg ccc 240 Pro Phe Arg Ile Val Glu AlaAla Cys Gly Met Lys Trp Ile Leu Pro 65 70 75 80 ttc atc ttc tgc ccc ctttct ggc ttc ctt ttc ttc acc acc atc tac 288 Phe Ile Phe Cys Pro Leu SerGly Phe Leu Phe Phe Thr Thr Ile Tyr 85 90 95 ctc acc tcc ctc ttc ctg atgacg gtg agc ata gaa cgt ttt ctg agc 336 Leu Thr Ser Leu Phe Leu Met ThrVal Ser Ile Glu Arg Phe Leu Ser 100 105 110 gta gcc tac cca ctg tgg tacaaa acc cgg ccc cgg ctg gcc cag gct 384 Val Ala Tyr Pro Leu Trp Tyr LysThr Arg Pro Arg Leu Ala Gln Ala 115 120 125 ggt ctg gtc agt ggc atc tgttgg ttc ttg gca tca gct cac tgt agt 432 Gly Leu Val Ser Gly Ile Cys TrpPhe Leu Ala Ser Ala His Cys Ser 130 135 140 gtg att tat gtc act gaa tactgg gga aat gca acc tac agc cag ggg 480 Val Ile Tyr Val Thr Glu Tyr TrpGly Asn Ala Thr Tyr Ser Gln Gly 145 150 155 160 acc aac gga acc tgc tacctg gaa ttc cgg gag gac cag ctg gcc atc 528 Thr Asn Gly Thr Cys Tyr LeuGlu Phe Arg Glu Asp Gln Leu Ala Ile 165 170 175 ctc ctc ccc gtg cga ctggaa atg gct gtg gtc ctt ttc atg gtg ccc 576 Leu Leu Pro Val Arg Leu GluMet Ala Val Val Leu Phe Met Val Pro 180 185 190 ctg tgt att acc agt tactgc tac agt cgc ctg gtg tgg att ctg agc 624 Leu Cys Ile Thr Ser Tyr CysTyr Ser Arg Leu Val Trp Ile Leu Ser 195 200 205 cag gga gcc agc cgg cgcagg cgc aag aga gtg atg ggg ctt ctt gta 672 Gln Gly Ala Ser Arg Arg ArgArg Lys Arg Val Met Gly Leu Leu Val 210 215 220 gcc acg ttg ctc atc ttcttt gtc tgc ttc ggc ccc tac aat atg tcc 720 Ala Thr Leu Leu Ile Phe PheVal Cys Phe Gly Pro Tyr Asn Met Ser 225 230 235 240 cac gtg gtg ggc tacgtg cgc ggt gag agt ccg acc tgg cgg agc tac 768 His Val Val Gly Tyr ValArg Gly Glu Ser Pro Thr Trp Arg Ser Tyr 245 250 255 gtg ctt ctc ctc agcacc ctc aac tct tgt att gac cct ctg gtt ttc 816 Val Leu Leu Leu Ser ThrLeu Asn Ser Cys Ile Asp Pro Leu Val Phe 260 265 270 tac ttt tca tcc tccaag ttc caa gcc gac ttt cat cag ctc ctg tct 864 Tyr Phe Ser Ser Ser LysPhe Gln Ala Asp Phe His Gln Leu Leu Ser 275 280 285 agg ctg atc aga gcttgt gtg cct tgg act cag gaa gtc agc ttg gaa 912 Arg Leu Ile Arg Ala CysVal Pro Trp Thr Gln Glu Val Ser Leu Glu 290 295 300 ctg aag gta aag aacgga gaa gag cca tcc aag gaa tgt ccg agc tag 960 Leu Lys Val Lys Asn GlyGlu Glu Pro Ser Lys Glu Cys Pro Ser 305 310 315 6 319 PRT Rattus sp. 6Met Asp Thr Ser Phe Phe Pro Gly Asn His Trp Leu Phe Phe Ser Val 1 5 1015 Asp Leu Leu Val Phe Leu Val Gly Leu Pro Leu Asn Val Met Ala Leu 20 2530 Val Val Phe Val Asn Lys Leu Arg Arg Arg Pro Val Ala Val Asp Leu 35 4045 Leu Leu Leu Asn Leu Thr Ile Ser Asp Leu Leu Leu Leu Leu Phe Leu 50 5560 Pro Phe Arg Ile Val Glu Ala Ala Cys Gly Met Lys Trp Ile Leu Pro 65 7075 80 Phe Ile Phe Cys Pro Leu Ser Gly Phe Leu Phe Phe Thr Thr Ile Tyr 8590 95 Leu Thr Ser Leu Phe Leu Met Thr Val Ser Ile Glu Arg Phe Leu Ser100 105 110 Val Ala Tyr Pro Leu Trp Tyr Lys Thr Arg Pro Arg Leu Ala GlnAla 115 120 125 Gly Leu Val Ser Gly Ile Cys Trp Phe Leu Ala Ser Ala HisCys Ser 130 135 140 Val Ile Tyr Val Thr Glu Tyr Trp Gly Asn Ala Thr TyrSer Gln Gly 145 150 155 160 Thr Asn Gly Thr Cys Tyr Leu Glu Phe Arg GluAsp Gln Leu Ala Ile 165 170 175 Leu Leu Pro Val Arg Leu Glu Met Ala ValVal Leu Phe Met Val Pro 180 185 190 Leu Cys Ile Thr Ser Tyr Cys Tyr SerArg Leu Val Trp Ile Leu Ser 195 200 205 Gln Gly Ala Ser Arg Arg Arg ArgLys Arg Val Met Gly Leu Leu Val 210 215 220 Ala Thr Leu Leu Ile Phe PheVal Cys Phe Gly Pro Tyr Asn Met Ser 225 230 235 240 His Val Val Gly TyrVal Arg Gly Glu Ser Pro Thr Trp Arg Ser Tyr 245 250 255 Val Leu Leu LeuSer Thr Leu Asn Ser Cys Ile Asp Pro Leu Val Phe 260 265 270 Tyr Phe SerSer Ser Lys Phe Gln Ala Asp Phe His Gln Leu Leu Ser 275 280 285 Arg LeuIle Arg Ala Cys Val Pro Trp Thr Gln Glu Val Ser Leu Glu 290 295 300 LeuLys Val Lys Asn Gly Glu Glu Pro Ser Lys Glu Cys Pro Ser 305 310 315 7 17DNA artificial sequence PCR primer 7 cattagcatc tgtgatg 17 8 20 DNAartificial sequence PCR primer 8 ctagctcgga cactccttgg 20 9 20 DNAartificial sequence PCR primer 9 gccatagcac tgagccaatg 20 10 20 DNAartificial sequence PCR primer 10 ttgtagccac gttgctcatc 20 11 27 DNAartificial sequence PCR primer 11 taggatccat ggacacaagc ttcttcc 27 12 29DNA artificial sequence PCR primer 12 tactcgagct agctcggaca ttccttgga 29

1. A method for identification of an agent that modulates activity ofG-protein coupled receptor 41 (GPR 41), or G-protein coupled receptor 42(GPR 42) which method comprises: (i) contacting a test agent with GPR41, GPR 42 or a variant of either thereof which is capable of couplingto a G-protein; and (ii) monitoring for GPR 41 or GPR 42 activity in thepresence of a G-protein; thereby determining whether the test agentmodulates GPR 41 or GPR 42 activity.
 2. A method according claim Iwherein the test agent is contacted in step (i) with cells that expressGPR 41, GPR 42 or a said variant of either thereof.
 3. A methodaccording to claim I wherein the test agent is contacted in step (i)with the membrane of cells that express GPR 41, GPR 42 or a said variantof either thereof.
 4. A method according to claim 2 or 3 wherein thecells are adipocytes.
 5. A method according to claim 4 wherein theadipocytes are provided as a differentiated cell line.
 6. A methodaccording to claim 4 wherein the adipocytes are primary adipocytesharvested from a human or animal donor.
 7. A method according to any oneof the preceding claims wherein the variant has at least 70% sequenceidentity to SEQ ID NO: 2 or SEQ ID NO:
 4. 8. A method according to anyone of the preceding claims wherein the G-protein is G_(i)-protein.
 9. Amethod according to claim 8 wherein step (ii) comprises determiningwhether G_(i)-protein is activated.
 10. A test kit suitable foridentification of an agent that modulates GPR 41 or GPR 42 activity,which kit comprises: (a) GPR 41, GPR 42 or a variant of either thereofwhich is capable of coupling to a G_(i)-protein; and (b) means formonitoring GPR 41 or GPR 42 activity.
 11. A kit according to claim 10wherein component (a) comprises cells which express GPR 41, GPR 42 or asaid variant of either thereof.
 12. A kit according to claims 10 or 11wherein component (b) comprises means for determining whetherG_(i)-protein is activated.
 13. A method for identification of an agentthat inhibits lipolysis, which method comprises contacting adipocytes invitro with a test agent identified by the method of any one of claims 1to 9 and monitoring lipolysis, thereby determining whether the testagent is an inhibitor of lipolysis.
 14. An activator of GPR 41 or GPR 42activity identified by a method according to any one of claims 1 or 9,an inhibitor of lipolysis identified by a method according to claim 13or a polynucleotide which encodes GPR 41, GPR 42 or a variantpolypeptide of either thereof as defined in claim 1, for use in a methodof treatment of the human or animal body by therapy.
 15. An activator,inhibitor or polynucleotide according to claim 14 for use in thetreatment of dyslipidaemia, coronary heart disease, atherosclerosis,thrombosis or obesity, angina, chronic renal failure, peripheralvascular disease, stroke, type II diabetes or metabolic syndrome(syndrome X).
 16. A polynucleotide according to claim 14 or 15comprising (a) the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 3,(b) a sequence which hybridizes under stringent conditions to thecomplement of SEQ ID NO: 1 or SEQ ID NO: 3, (c) a sequence that isdegenerate as a result of the genetic code with respect to a sequencedefined in (a) or (b), or (d) a sequence having at least 60% identity toa sequence as defined (a), (b) or (c).
 17. Use of an activator,inhibitor or polynucleotide as defined in claim 14 in the manufacture ofa medicament for the treatment of dyslipidaemia, coronary heart disease,atherosclerosis, thrombosis or obesity, angina, chronic renal failure,peripheral vascular disease, stroke, type II diabetes or metabolicsyndrome (syndrome X).
 18. An activator of GPR 41 or GPR 42 activity ora polynucleotide which encodes GPR 41, GPR 42 or a variant polypeptideof either thereof as defined in claim 1, for use in the treatment ofdyslipidaemia, coronary heart disease, atherosclerosis, thrombosis orobesity, angina, chronic renal failure, penpteral; vascular disease,stroke, type II diabetes or metabolic syndrome (syndrome X).